Oxygen plays a critical biological role as the terminal electron acceptor in the mitochondria of vertebrate cells. During evolution, these cells have developed ways to sense alterations in oxygen levels and, during this process, acquired the ability to conditionally modulate the expression of genes involved in adaptive physiological responses to hypoxia including angiogenesis, erythropoiesis, and glycolysis. These genes include vascular endothelial growth factor, eryhtropoietin, several glycolytic enzymes and inducible nitric oxide synthase, and have all been shown to contain hypoxia responsive elements (HREs). Under hypoxic conditions these response elements are recognized by a heterodimeric complex consisting of the hypoxia inducible factor-1a (HIF-1a) and Arnt. Both these transcription factors belong to the rapidly growing family of basic-helix-loop-helix (bHLH)-PAS (Per, Arnt, Sim) proteins.
A family of helix-loop-helix proteins designated Id has been identified as antagonists of bHLH transcriptional regulators. bHLH proteins typically bind regulatory sequences in a heterodimeric configuration and function to activate differentiation-linked gene expression. The heterodimer usually comprises a class A bHLH protein together with a class B bHLH protein. In the presence of excess Id protein, the class A bHLH partner is typically titrated out through heterodimerization with Id protein.
Dysregulation or overfunction of HIF-1 a might cause a variety of pathological conditions including tumor progression and inflammatory angiogenesis. Consequently, there is a need for identification of compounds acting as negative regulators of HIF-1a, said compounds being potentially useful against medical conditions related to angiogenesis and tumor progression.
Primary culture of mouse cornea epithelium cells were transfected with either antisense IPAS expression plasmid or empty vector (vector) and incubated under normoxic (N, 21% O2) or hypoxic (H, 1% O2) conditions for 24 h. Total RNAs from the cells were extracted and Northern blot analysis using radiolabelled mouse VEGF cDNA probe was performed. Total RNAs form normoxic and hypoxic Hepa1c1c7 cells were supplied as a reference for VEGF induction. As a loading control, 18S RNA levels are shown.